RAD59 is required for efficient repair of simultaneous double-strand breaks resulting in translocations in Saccharomyces cerevisiae

Abstract

Exposure to ionizing radiation results in a variety of genome rearrangements that have been linked to tumor formation. Many of these rearrangements are thought to arise from the repair of double-strand breaks (DSBs) by several mechanisms, including homologous recombination (HR) between repetitive sequences dispersed throughout the genome. Doses of radiation sufficient to create DSBs in or near multiple repetitive elements simultaneously could initiate single-strand annealing (SSA), a highly efficient, though mutagenic, mode of DSB repair. We have investigated the genetic control of the formation of translocations that occur spontaneously and those that form after the generation of DSBs adjacent to homologous sequences on two, non-homologous chromosomes in Saccharomyces cerevisiae. We found that mutations in a variety of DNA repair genes have distinct effects on break-stimulated translocation. Furthermore, the genetic requirements for repair using 300 bp and 60 bp recombination substrates were different, suggesting that the SSA apparatus may be altered in response to changing substrate lengths. Notably, RAD59 was found to play a particularly significant role in recombination between the short substrates that was partially independent of that of RAD52. The high frequency of these events suggests that SSA may be an important mechanism of genome rearrangement following acute radiation exposure.

Introduction

Acute doses of ionizing radiation, either through accidental [1], [2] or therapeutic [3] exposure, have been shown to generate a diverse spectrum of genomic aberrations, with the amount of DNA damage increasing with increases in the radiation dose [4], [5], [6]. This damage includes double-strand breaks (DSBs) that need to be repaired for a cell to survive [7], [8]. Repair can occur either through pathways that utilize HR between homologous sequences or through mechanisms that require little to no homology [9], [10], [11]. Following exposure to ionizing radiation, strains of the yeast Saccharomyces cerevisiae that lack proteins in the Rad52 epistasis group inefficiently repair DSBs and display significant loss of viability [7], [12], [13], emphasizing the importance of the HR pathway for radiation resistance. In contrast, cells lacking components of the non-homologous end-joining (NHEJ) pathway do not exhibit this sensitivity [11]. The HR and NHEJ apparatuses in yeast have homologous counterparts in higher eukaryotes, where both pathways appear important for radiation resistance [11], [14], [15], [16], [17].

Following the creation of a DSB, homologous sequence is sought out, typically from the sister chromatid or homologous chromosome, for use as a template for repair [10]. It has been shown that the HR machinery can also utilize homologous sequences located on non-homologous chromosomes [18], [19]. Since evidence of such promiscuous interactions appear infrequently in normal cells it is clear that there are mechanisms to prevent them, thus maintaining the integrity of the genetic material. In cancerous and diseased cells, however, translocations, insertions, deletions, duplications, and truncations are frequently observed [20], [21], [22] suggesting that these mechanisms have been abrogated such that inappropriate templates are utilized for repair.

The numerous repetitive elements dispersed throughout all eukaryotic genomes are likely candidates for these inappropriate templates. In S. cerevisiae, there are over 100 copies of the 250 bp delta repeat [23], [24]. Spontaneous recombination events between these and other repetitive elements have been shown to lead to genome rearrangements [25], [26], [27]. In humans, the 300 bp Alu repeats are the most abundant repetitive element with approximately 10⁶ copies identified to date. These repeats are spaced an average of 4 kb apart, with each one possessing from 70% to 98% homology to a consensus sequence [28]. Evidence is accumulating that these repeats are involved in the genomic instability associated with some diseases [21], [29]. Previous studies have examined HR involving substrates that are similar in size to these repetitive elements and have shown that translocations can occur spontaneously, while DSBs created at one or both substrates greatly increase their frequency [19], [30], [31], [32].

In this study, we have monitored repair following the generation of DSBs at multiple loci in diploid S. cerevisiae cells, as might occur following exposure to ionizing radiation, and found a physical translocation involving homologous sequences on two chromosomes to be the major product. The frequency at which this translocation is recovered, the non-conservative nature of the recombination event, and the genetic requirements for recovery have implicated single-strand annealing (SSA) [33], [34], a highly efficient form of HR. Furthermore, we have found that the genetic control of SSA varies with the lengths of the substrates, suggesting that the amount of homology available for repair may determine the nature of the apparatus that is engaged. Most notably, Rad59 appears to play a particularly important role in driving translocation formation with short substrates. The function of Rad59 in translocation formation is distinct from that of its paralog [35], the central homologous recombination protein Rad52, suggesting that Rad59 can act in multiple contexts in HR.